A lncRNA Dleu2-encoded peptide relieves autoimmunity by facilitating Smad3-mediated Treg induction

Micropeptides encoded by short open reading frames (sORFs) within long noncoding RNAs (lncRNAs) are beginning to be discovered and characterized as regulators of biological and pathological processes. Here, we find that lncRNA Dleu2 encodes a 17-amino-acid micropeptide, which we name Dleu2-17aa, that is abundantly expressed in T cells. Dleu2-17aa promotes inducible regulatory T (iTreg) cell generation by interacting with SMAD Family Member 3 (Smad3) and enhancing its binding to the Foxp3 conserved non-coding DNA sequence 1 (CNS1) region. Importantly, the genetic deletion of Dleu2-17aa in mice by start codon mutation impairs iTreg generation and worsens experimental autoimmune encephalomyelitis (EAE). Conversely, the exogenous supplementation of Dleu2-17aa relieves EAE. Our findings demonstrate an indispensable role of Dleu2-17aa in maintaining immune homeostasis and suggest therapeutic applications for this peptide in treating autoimmune diseases.

The authors tried to show that LncRNA Dleu2-Encoding Peptide attenuates EAE by facilitating Smad3-mediated Treg induction.They showed that lncRNA Dleu2 encoded Dleu2-17aa, which promoted inducible regulatory T (iTreg) cell generation through interacting with SMAD Family Member 3 (Smad3) and enhancing the binding of Smad3 to the Foxp3 conserved non-coding DNA sequence 1 35 (CNS1) region.Moreover, they showed that genetic deletion of Dleu2-17aa in mice through start codon mutation impaired iTreg generation and exacerbated EAE, and that exogenously supplemented Dleu2-17aa attenuated EAE.The manuscript is well written.
The topic is important.However, the results of EAE experiments are premature and not convincing.There are several major concerns.
1. Figure 5 B shows extremely severe demyelination in scrPEP-treated control EAE mice.However, Figure 7C shows mild demyelination in WT control EAE mice, and moderate demyelination in Dleu2-17aa KO mice with EAE.In fact, there is much more severe demyelination in scrPEP-treated control EAE mice than Dleu2-17aa KO mice with EAE.How to explain this very confusing result?2. The authors should perform experiments (such as in vitro recall assay) to determine whether Dleu2-17aa KO or exogenously supplemented Dleu2-17aa affect T cell priming and reactivation in the EAE model.3. The authors used global Dleu2-17aa KO or exogenously supplemented Dleu2-17aa.Does Dleu2-17aa express in neurons and glia (oligodendrocytes, neurons, microglia, and astrocytes)?All these cell types play critical roles in the pathogenesis of EAE.Does global Dleu2-17aa KO or exogenously supplemented Dleu2-17aa affects the activity of microglia, and astrocytes during EAE?Does global Dleu2-17aa KO or exogenously supplemented Dleu2-17aa affects the viability of neurons or oligodendrocytes? 4. The authors should quantify inflammation, demyelination, oligodendrocyte loss, and axon degeneration in the CNS of EAE models.
-They first provide experimental evidences that Dleu2 encodes a 17-amino-acid micropeptide.
-The micropeptide is abundantly expressed in T cells -It promoted inducible regulatory T (iTreg) cell generation in vitro -Mechanistically, they show that Dleu2 micropeptide interact with SMAD Family Member 3 (Smad3) and this results in an increase of the binding of Smad3 to a promoter region of the Foxp3 gene.
-This consequently result in a better activation of the Foxp3 target genes upon TGFβ activation.
-They performed start codon mutation to generate KO of this peptide in mice.Its mutation compromised iTreg generation and exacerbated experimental autoimmune encephalomyelitis (EAE).
The study is interesting and is in the cope of EMBO reports.I am not specialist of Treg cells biology but the experiments provided look convincing, their conclusions correctly supported.The Dleu2 peptide characterization is well done and the data are convincing.
I have therefore no special concerns regarding the manuscript publication.
Other points: -English grammar (tenses and conjugations) should be checked -MS data must be made available.As well, an excel file describing each protein identified must be provided.
-I have also one suggestion to strengthen the story.I am wondering whether the Dleu2 peptide enhance Smad binding only on the Foxp3 DNA region.Is this specific of this sequence or is the Smad binding increased on all binding siytes targeted?Are some adjacent nucleotides involved?Using a synthetic Smad3 consensus-binding site (or binding from other promoters/enhancers) and testing if addition of the Dleu2 peptide also enhances the Smad3 binding can test this.
-lane 160.The other interactors should be described and listed (how many, names etc) -lane 222.Typo; "its" but not "tis" -------------Referee #3: The authors identified a novel 17aa short peptide encoded by Dleu2, a previously designated lncRNA, that is important for Smad3 binding to the CNS1 enhancer of Foxp3 locus.Mice treated with Dleu2 induced more Tregs during EAE and had better outcome, while deletion of Dleu2 resulted in impaired Treg induction and worse clinical scores.The studied is elegantly designed and well performed with a novel discovery, however, there are certain issues with how data was presented and the quality of the figures, and here are the specifics: 1.In Figure 2D, the representative plots are all selected from the samples with the lowest percentage.Especially for 5uM and 10uM groups, the "representative plots" can even be considered outliers.a.While treating cells with the small peptide has therapeutic implications, dose dependent uptake needs to be demonstrated.b.Maybe over expressing Dleu2 in cells and compare low expressers and high expressers for their Foxp3 induction potential could be a better experiment to perform.4B, what cis element is used and in what cell type?If the main focus of this manuscript is on CNS1, should a CNS1 reporter be used in this experiment?3.In Figure 6G, Treg population in barrier tissues should be shown and their expression of RORgt analyzed.pTregs are especially abundant in the gut, where RORgt expression marks the microbiota induced pTregs.CNS1 deficient mice have been shown to harbor fewer Tregs in the intestine.

RESPONSE TO THE REVIEWERS' COMMENTS
Changes in the revised manuscript text are underlined.
The topic is important.However, the results of EAE experiments are premature and not convincing.There are several major concerns.Comment 1. Figure 5 B shows extremely severe demyelination in scrPEP-treated control EAE mice.However, Figure 7C shows mild demyelination in WT control EAE mice, and moderate demyelination in Dleu2-17aa KO mice with EAE.In fact, there is much more severe demyelination in scrPEP-treated control EAE mice than Dleu2-17aa KO mice with EAE.How to explain this very confusing result?Re: We are deeply grateful to the reviewer for catching this inappropriate image selection.We thoroughly re-examined the original images and provided pictures consistent with the statistics of demyelination.(revised Fig. 5C, 7C) Here we present more original LFB staining images of these experiments.Re: We sincerely appreciate the valuable suggestion provided by the reviewer.Following the reviewer's suggestion, we performed in vitro T cell recall assay.Specifically, splenic CD4 + T cells were isolated from WT and KO mice following EAE induction.Then the CD4 + T cells were re-stimulated with the myelin oligodendrocyte glycoprotein (MOG) 33-35 peptide in the presence of irradiated antigen presenting cells from Rag1 -/-mice spleen.Using this approach, we measured cytokine production by intracellular staining and flow cytometric analysis.No differences were observed in the frequencies of IL-17A + or IFN-γ + CD4 + T cells between WT and KO groups (revised Appendix Fig. S7A).Proliferative capacity was also assessed and found to be comparable between two groups upon MOG rechallenge, as demonstrated by cell trace violet dilution assays (revised Appendix Fig. S7B).These implied Dleu2-17aa modulates EAE pathogenesis by regulating Treg induction rather than T cell reactivation.
Comment 3. The authors used global Dleu2-17aa KO or exogenously supplemented Dleu2-17aa.Does Dleu2-17aa express in neurons and glia (oligodendrocytes, neurons, microglia, and astrocytes)?All these cell types play critical roles in the pathogenesis of EAE.Does global Dleu2-17aa KO or exogenously supplemented Dleu2-17aa affects the activity of microglia, and astrocytes during EAE?Does global Dleu2-17aa KO or exogenously supplemented Dleu2-17aa affects the viability of neurons or oligodendrocytes?
Re: We would like to express our sincere gratitude to the reviewer's thoughtful suggestion to examine Dleu2-17aa localization within the CNS.Following the reviewer's guidance, we conducted immunofluorescent co-staining experiments to evaluate colocalization of Dleu2-17aa with various cell types, including CD4 + T cells, microglia, astrocytes, and neurons in spinal cord sections from Dleu2-17aa and scrambled peptide-treated EAE mice.The results revealed Dleu2-17aa is expressed in almost all the CD4 + T cell.Furthermore, we observed some colocalization with microglia, limited colocalization with astrocytes, and minimal colocalization with neurons.While supplementation of Dleu2-17aa conferred some protective effects, it did not substantially alter activity or viability of glia or neurons, as the morphology did not change (revised Appendix Fig. S6).However, we did observe reduced CD4 + T cell infiltration in the spinal cords of Dleu2-17aa-treated EAE mice compared with scrPEP controls.We extend our heartfelt thanks to the reviewer for the valuable time and expertise in reviewing our manuscript and for this analysis that broadens CNS characterization.
Comment 4. The authors should quantify inflammation, demyelination, oligodendrocyte loss, and axon degeneration in the CNS of EAE models.
Re: We appreciate the reviewer's insightful suggestion.As advised, we quantified inflammatory foci and demyelinated lesions in the CNS using scoring systems, which are commonly used and well-established parameters in EAE research (revised Figures 7D and 5D).
The data revealed increased pathology in KO and WT and Dleu2-17aa supplemented EAE mice (revised Figures 7D and 5D).We are grateful to the reviewer for recommending these important analyses.
-They first provide experimental evidences that Dleu2 encodes a 17-amino-acid micropeptide.
-The micropeptide is abundantly expressed in T cells -It promoted inducible regulatory T (iTreg) cell generation in vitro -Mechanistically, they show that Dleu2 micropeptide interact with SMAD Family Member 3 (Smad3) and this results in an increase of the binding of Smad3 to a promoter region of the Foxp3 gene.
-This consequently result in a better activation of the Foxp3 target genes upon TGFβ activation.
-They performed start codon mutation to generate KO of this peptide in mice.Its mutation compromised iTreg generation and exacerbated experimental autoimmune encephalomyelitis (EAE).
The study is interesting and is in the cope of EMBO reports.I am not specialist of Treg cells biology but the experiments provided look convincing, their conclusions correctly supported.The noting the plots in Figure 2D were not fully representative.We have updated the figure with more typical examples from each dose group.
We would like to sincerely thank the reviewer for raising these important issues as demonstrating dose-dependent uptake of the Dleu2-17aa helps strengthen our conclusions.In the revised manuscript, we showed dose-dependent penetration of fluorescence labeled Dleu2-17aa by CD4 + T cells, with maximal uptake observed between 25-50 μM (revised Fig. EV1B and C).
Additionally, as suggested, we have performed new experiments overexpressing Dleu2-17aa in T cells using a lentiviral vector.We sorted cells into medium and high Dleu2-17aa-expressing populations and induced iTreg differentiation.We found that high Dleu2-17aa-expressing T cells showed increased iTreg differentiation compared with low expressing T cells (revised Appendix Fig S3).
Comment 2. In Figure 4B, what cis element is used and in what cell type?If the main focus of this manuscript is on CNS1, should a CNS1 reporter be used in this experiment?
Re: We are extremely grateful to the reviewer for pointing out our oversight.In the revised manuscript, we have clarified the details of the luciferase reporter assays evaluating the effect of Dleu2-17aa on Smad3-mediated transcription.Specifically, we constructed a luciferase reporter plasmid containing the Foxp3 CNS1 region and transfected this reporter into NIH 3T3 cells along with a Smad3 expression vector.We observed that treatment with Dleu2-17aa led to a significant increase in CNS1-dependent luciferase expression compared with control.This indicated that Dleu2-17aa can enhance Smad3 transcription ability on the CNS1 element.Re: We are grateful to the reviewer for the constructive feedback to help us enhance our work.Following the reviewer's valuable suggestion, we examined the pTreg population and RORγt expression in the colon lamina propria of WT and Dleu2-17aa KO mice, as this tissue harbors abundant RORγt + pTregs.Using flow cytometry, we quantified Foxp3 + RORγt + double positive pTregs in the colonic lamina propria lymphocytes from WT and KO mice.We detected comparable frequencies of RORγt + pTregs between the two group (revised Figure 6I, J), indicating that Dleu2-17aa deficiency does not alter RORγt + pTreg homeostasis in the colon under steady state.
Comment 4. In addition to EAE, it would also be helpful to show if there is a difference between WT and KO animals in pTreg induction under homeostatic conditions, i.e in response to dietary antigen with OVA feeding etc.
Re: We appreciate the reviewer for taking the time to provide such thoughtful comments and suggestions.Following the reviewer's kind advise, we examined pTreg induction of WT and KO mice under homeostatic conditions by using an oral ovalbumin (OVA) feeding model.We fed WT and KO mice with OVA antigen plus cholera toxin subunit B (CTB) as adjuvant.No weight loss was observed in OVA and CTB fed mice (revised Fig. EV5A).We then analyzed Foxp3 + RORγt + pTregs in the colonic lamina propria lymphocytes.With flow cytometry (revised Fig. EV5B) and quantification (revised Fig. EV5C-E), we observed that Dleu2-17aa KO mice showed a significant reduction in the frequency of pTregs compared with WT mice.This suggested that Dleu2-17aa promotes pTreg differentiation driven by dietary antigens.The impaired oral antigen-induced pTreg formation aligns with the exacerbated EAE phenotypes observed in Dleu2-17aa KO mice.Taken together, these data revealed an important function for Dleu2-17aa in pTreg generation upon dietary antigen stimulation, complementing its effect on iTregs driven by TGF-β signaling.We thank the reviewer for this invaluable suggestion to assess pTregs under physiological conditions, which uncovered a novel role for Dleu2-17aa in oral tolerance and mucosal pTreg responses.Figures 5E.After the reviewer pointed this out, we realized the antibody markers in the flow plots were mistakenly swapped in Figure 5E.We have checked and replaced the Figure 5E with correct cytokine antibody label in the revised manuscript.We are extremely grateful to the reviewer for catching this issue and helping us re-evaluating our data to generate reliable data quality that will substantially strengthen the manuscript.Here we present original flow cytometry data in figure5E.

28th Nov 2023 1st Revision -Editorial Decision
Dear Prof. Wang, Thank you for the submission of your revised manuscript to our editorial offices.I have now received the reports from the three referees that I asked to re-evaluate your study, you will find below.As you will see, the referees now support the publication of the study in EMBO reports.Referees #1 and #3 have some remaining concerns or suggestions to improve the manuscript I ask you to address in a final revised manuscript.
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The authors should perform experiments (such as in vitro recall assay) to determine whether Dleu2-17aa KO or exogenously supplemented Dleu2-17aa affect T cell priming and reactivation in the EAE model.
Comment 3. In Figure6G, Treg population in barrier tissues should be shown and their expression of RORgt analyzed.pTregs are especially abundant in the gut, where RORgt expression marks the microbiota induced pTregs.CNS1 deficient mice have been shown to harbor fewer Tregs in the intestine.

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Are Figures 5E and 7E done in a similar fashion?The cytokine production profile (IFNg production and IL-17 production) looks drastically different between the two experiments.It might affect how the data is interpretated.
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